STUDYING THE ROLE OF THE DIVALENT METAL CATION ACTIVATOR OF THE CAMBIALISTIC ENZYME MANDELATE RACEMASE
Abstract
Mandalate racemase (MR) from Pseudomonas putida requires a divalent metal cation,
usually Mg2+, to catalyze the interconversion of the enantiomers of mandelate. Typical of
a cambialistic enzyme, the active site Mg2+ may be replaced by other metal ions.
Replacement by Co2+, Ni2+, and Mn2+ does not significantly alter the kinetic parameters
Kmapp, kcatapp, and (kcat/Km)app for the substrates (R)– and (S)–mandelate, and the alternative
substrate (S)–trifluorolactate. The competitive inhibition constants (Ki) for inhibition of
each metalloenzyme by benzohydroxamate did not vary significantly with the identity of
the metal ion; however, marked variation of the stability constant (K1) for metal ion–BzH
complex formation in solution was observed. Thus the enzyme appears to modify the
properties of the bound metal ion so that the catalytic roles of the different metal ions are
relatively invariant compared to their properties in solution (‘Hephaestus effect’). The
slight reduction in catalytic efficiency by Mn2+–MR was studied by examining the
dependence of kcatapp and (kcat/Km)app for Mn2+–MR on the solvent microviscosity. These
studies showed that the metal ion plays a role in the uniform binding of the transition
states for substrate/product association/dissociation and the chemical step.